1. Field of the Invention
This invention relates to the fields of virology and immunology. Particularly, but not exclusively, it relates to a method of inducing an immune response to HIV using a psoralen inactivated composition of HIV and a substance for achieving the same.
2. Description of the Related Art
Human Immunodeficiency Virus
Human Immunodeficiency Virus (HIV) is a retrovirus within the slow or Lentivirus group, and is the cause of Acquired Immunodeficiency Syndrome (AIDS). Some retroviruses that attack the immune system, such as HIV-1, are variable and mutate readily, creating many strains of varying genetic composition that hamper efforts to develop effective treatment. These strains, which may be categorized into groups or subtypes, have individual biological characteristics. Sequences within a subtype may have genetic clustering or similarities that sometimes reveal their common lineage. However, variations in evolutionary rate can produce differences among mutations even within a subtype. Further, the tendency of retroviruses to recombine with related retroviruses complicate the viral genotype.
HIV uses its RNA as a template for making complementary viral DNA in target cells through reverse transcription, viral DNA can then integrate into the DNA of an infected host. HIV infects cells having surface CD4, such as lymphocytes, monocytes, dendritic cells and macrophages, and destroys CD4 positive helper T lymphocytes. This process relies in part on two glycoproteins of HIV. These glycoproteins are gp120 (an Env glycoprotein, the exterior receptor-binding component) and a non-covalently interacting partner, gp41 (the Env transmembrane glycoprotein.) Gp120 and gp41 are associated in a trimeric unit, where three molecules of gp120 are exposed on the virion surface and are associated with three molecules of gp41 in the viral lipid membrane. Gp120 binds to a CD4 receptor on the surface of helper T cells. This binding is generally considered to be high affinity, and can be further enhanced by high sialic acid content on the surface of the virus; sialic acid reduces the threshold binding energy needed to overcome repulsive electrostatic forces. The virus then begins to fuse with the T cell, producing structural or conformational changes and exposing other receptors. Upon fusion, the gp120 fragment is shed, exposing the gp41 ectodomain in a process that also varies conformationally. Gp41 is then available to project peptide fusion domains for binding to the target cell. This leads to HIV entering and infecting the target cell.
The envelope of HIV begins formation from the plasma membrane of the host cell when the virus buds through the cell membrane. Thus, the envelope includes the lipid and protein constituents of the host cell. (Frank, Ines, Heribert Stoiber, et al., Vol. 10, pp. 1611-20 (1996)) (Stoiber, Heribert, et al., Vol. 15, pp. 649-74 (1997)) Some enveloped viruses use spike proteins to mimic the host molecules in order to bind to target cell receptors and to enter other target cells. However, these spikes can also be antigenic surfaces for immune system recognition. Yet HIV offers protection. In addition to the variability of conformational changes, gp120 provides other surface features that disguise it from immune detection and attack, such as a coating of glycoproteins, covalently bound sialic acid residues, or steric occlusion. (Haurum, John, Treffen Thiel, et al., Vol. 7(10), pp. 1307-13 (October 1993)) (Sande, Merle, et al., The Medical Management of Aids, (6th ed. 1999)) (Cohen, P. T., The AIDS Knowledge Base, (3rd ed. February 1999))
The core of the HIV virion functions as a command center. Inside an HIV virion is a capsid composed of the viral protein, p24 (CA). The capsid holds two single strands of RNA, each strand of which provides a copy of HIV's nine genes, which encode 15 proteins. Of the nine genes, three (gag, pol and env) are considered essential. Six additional genes are also found within the 9-kilobase pair RNA genome (vif, vpu, vpr, tat, rev and nef) More specifically, the env gene holds the information or code for creation of gp160, which breaks down into gp120 and gp41. Likewise the gag gene encodes the matrix (p17 or MA), capsid (p24 or CA), nucleocapsid (p9 or NC) and p6. The pol gene provides the genetic information for the virus to produce the reverse transcriptase enzyme as well as the integrase enzyme and RNAse H enzyme. The other six genes are regulatory, and control the mechanisms of infection and replication (vif, vpu, vpr, tat, rev and nef). Among other things, the nef gene holds information for efficient replication, while vpu holds information regulating the release of new viral particles from the infected host cell. Ultimately, in order for HIV to infect a target cell, it must inject the HIV genetic material into the target cells cytoplasm.
As noted above, the nef gene is believed to aid efficient replication of HIV. The creation of a new virus particle occurs at the host cell's membrane. Nef appears to affect an infected cell's environment in a way that optimizes replication. Viral proteins collect near the host cells membrane, bud out within the membrane, and break away. These proteins are the three structural proteins (gp160, gp120, gp41) plus two other internal precursor polyproteins (Gag and the Gag-Pol). The Gag-Pol protein brings two strands of the positive RNA into the bud, while protease cuts itself free. After the virus has budded, protease cuts itself free and cuts up the rest of the proteins in Gag or Gag-Pol, releasing the various structural proteins and reverse transcriptase. The viral proteins are not functional until they are separated by the protease. Thus, protease is responsible for cleavage of Gag-Pol and the smaller Gag polyprotein into structural proteins. Released proteins p24, p7 and p6 form a new capsid, while at the base of the lipid membrane is p24. In this process, gp160 breaks down into gp120 and gp41 by a host enzyme.
Most HIV vaccines use portions of the envelopes of these glycoproteins (gp160, gp120, and gp41) in an attempt to induce production of neutralizing antibodies against the envelope spikes of the virus. (Johnston, et al., 2001) Some have been successful in producing high titers of neutralizing antibodies. The thought behind this approach is that the antibodies that bind to these glycoproteins would neutralize the virus and prevent infection. A functioning immune system could then activate the complement system, which would cascade to lysis and destroy the virus. The complement system is a series of circulating proteins that “complements” the role of antibodies. The components of the complement system are activated in sequence or turn, which is the complement cascade. The conclusion of complement is a protein complex, the Membrane Attack Complex (MAC) that seeks to attach to an invading organism's surface and to destroy it by puncturing its cell membrane.
However, HIV provides an additional protection against humoral immune response. HIV will activate human complement systems even in the absence of specific antibodies. This activation would be harmful to the virus if complement were left unimpeded to reach MAC, triggering virolysis. However, HIV avoids virolysis by incorporating into its structure various molecules (e.g., CD55, CD59) that regulate complement. HIV includes these cell membrane molecules in the virus membrane upon budding from infected cells, or by attachment to gp41 and gp120. Complement Factor H may be incorporated into the structures of both gp41 and gp120. Factor H inhibits the activity of C3b, a molecule that is central within complement cascade. This interaction with complement components enables HIV to take advantage of complement activation to enhance infectivity, follicular localization, and target cell range.
Vaccine Therapy and Related Art
Immunotherapy involves the use or stimulation of the immune system with respect to a condition or sensitivity. Vaccines are a form of immunotherapy. In 1955, Dr. Salk introduced the poliovirus vaccine; this vaccine used the chemical formaldehyde (formalin) to kill the virus or render it non-infective or inactive, so that it could be administered to patients. In 1961 Dr. Sabin introduced a live attenuated relatively avirulent poliovirus vaccine. The Sabin vaccine was basically composed of viral mutants capable of eliciting an immune response but not capable of significant active replication or virulence, and therefore were considered relatively safe for human use.
There have been effective vaccines against retroviruses in animals. One vaccine is available for feline immunodeficiency retrovirus (or FIV) (i.e., Fel-O-Vax); a second example is a vaccine against Equine Infectious Anemia Virus (or EIAV), (i.e., EIAV(UK)deltaS2) an important retroviral infection of horses. These vaccines argue powerfully that vaccines can work against retroviruses, although neither disease is an ideal model for HIV in humans. (Beyer, 2003)
However, a vaccine for HIV has proven elusive. The vast majority of vaccines under consideration, research, or trials are comprised of either “live” attenuated viral particles or whole inactivated viral particles. The use and research of recombinant technology, adenoviral vectors, DNA-based vaccines or a combination thereof has tested the boundaries of immunology, offering some hopes for addressing HIV. Such immunogenic compositions could be used for the following purposes:
To enhance the immune system of a person who has already been infected with the disease systemically.
To prevent a person from contracting the disease after exposure.
To prevent a person from contracting the disease prior to exposure. This is the most common use for a vaccine today.
To prevent a patient from contracting a different strain of HIV disease, particularly non-compliant or immunosuppressed patients.
To prevent vertical transmission from mother to fetus or from mother to newborn.
To attenuate HIV disease in an HIV negative patient who contracts the disease at a later date
To research potential compositions and methods for any of the purposes above
Unfortunately, medicine lacks a definition for HIV immunity. (Gonsalves, Gregg, Basic Science (2000)) (Cohen, 1999) This is a fundamental problem with an important consequence: there is no known correlate of protection against HIV. However, there are well-characterized correlates for disease progression, such as viral loads and CD4 counts. Furthermore, there is no evidence that any of the current candidate vaccines can elicit responses in HIV-positive patients that would improve these parameters (viral loads and CD4 counts) for an extended period. (Beyrer, Chris, “The HIV/AIDS Vaccine Research: An Update.” The Hopkins Report (January 2003)) Additionally, while there have been advances in some animal models, there is no validated animal model system for testing vaccine candidates, an obvious limitation when working with a high fatality pathogen such as HIV. (Beyrer, 2003) Current life expectancy after contracting HIV disease is approximately 10 to 15 years. Even a vaccine that failed to prevent transmission but extended life expectancy of a patient after contracting the disease would constitute an improvement.
Inactivated viruses may be useful for research and medicine. In fact, most of the successful early vaccines relied on inactivated virus. Inactivation produces a virus that is not infective, yet still induces an immune response based on its residual characteristics. An inactivated virus is typically generated from stocks of a virulent strain grown in cultured cells or animals. A potentially virulent virus is then made non-infectious or inactivated by chemical treatment. Viruses are by definition non-viable entities; they do not consume oxygen and food, nor do they produce waste; they replicate via their host, as described above for HIV. Viruses have no inherent metabolic activity and do not produce adenosine triphosphate (ATP). However, a live virus vaccine is capable of reproduction, while a killed virus vaccine is not. In general, live vaccines are more efficacious but also more dangerous than killed vaccines.
When a virus is inactivated, an immunogenic composition based on inactivated virus must retain its antigenicity in order to be useful. The inactivation process should preserve the three-dimensional structure of the virus while at the same time eliminating its virulence. Many methods are available to inactivate or kill a virus, but most destroy or change the three-dimensional structure of the virion, harming its antigenic characteristics. Originally, formaldehyde (formalin) treatment was used; for example, the Salk poliovirus vaccine was a formalin-inactivated preparation of three virus serotypes. Despite its wide use in early vaccines, formalin is difficult to remove and therefore poses the danger of residual toxicity. More recently, β-propriolactone has become a commonly used chemical to inactive a virus because residual amounts of the reagent can be readily hydrolyzed into non-toxic products. U.S. Pat. No. 4,169,204 to J. Hearst, et al., suggested the use of psoralens with irradiation to inactivate viruses for vaccine preparation. Psoralens are attractive because of their ability to inactivate virus without damaging the structure and without harmful residue. (Hanson, C. V., Bloodcells, Vol. 18(1), pp. 7-25 (1992)) Psoralens occur naturally in plants, including limes and celery, which use them to attack insects and fungi.
As noted above, the general notion of using psoralen to inactivate viruses is known. For example, U.S. Pat. No. 5,106,619 disclosed psoralen inactivation of a live virus in order to prepare vaccines. That invention involved treatment or inactivation of virions using furocoumarins, including 4′-aminomethyl-4,5′, 8-trimethylpsoralen hydrochloride (AMT), and ultraviolet light in a limited oxygen environment. The inactivation is directed to double and single stranded DNA viruses, double and single stranded RNA viruses, and enveloped and non-enveloped viruses. This disclosure was general, and did not specifically contemplate HIV.
Some inventors have contemplated the use of psoralen in an HIV vaccine or composition. U.S. Pat. No. 6,107,543 disclosed a whole particle HIV immunogen that is inactivated preferably by gamma radiation; also disclosed, however, are a variety of alternative inactivation methods including psoralen, formalin, β-propriolactone, etc. The whole particle is treated for removal of the outer envelope proteins gp120 or gp160, while retaining the remainder of the structure. An alternative embodiment is a reduced immunogen comprising the remaining purified gene products, such as those encoded by the gag genes, the pol genes, the trans-membrane protein gp41, or the remaining genes of the HIV genome.
U.S. Pat. Nos. 6,383,806 and 6,503,753 disclosed a composition and method for development of an HIV vaccine based on psoralen photoinactivation of Reverse Transcriptase (RT). In other words, the objective of this invention is to promote an immune response based on the inactivation of a single inactivated enzyme within HIV. Preservation of the remainder of the particle is deemed to enhance immune response to the composition.
Although psoralen has been contemplated by inventors for use in an HIV immunogen or vaccine, none have looked to certain structural preservation issues inherent with psoralen inactivation of HIV. For example, HIV is highly mutagenic, changing structures frequently in the process of reverse transcription. Mutation may provide a means for an HIV strain to escape immune response caused by a vaccine. (Cohen, 1999) In addition, the preservation of HIV structure may result in the preservation of HIV components that disadvantage immune response.
Past efforts have not focused on the problems of mutation. HIV is a highly mutagenic retrovirus which, through reverse transcriptase converts its RNA into DNA. HIV reverse transcriptase is error prone, leading to mutation. Further, rapid replication exacerbates mutation. The high level of genomic diversity in HIV complicates diagnosis, treatment, and public health monitoring of disease progression. In particular, this diversity is manifested in biological peculiarities characterizing as infectivity, transmissibility, and immunogenicity. The divergence in viral genotypes of HIV has contributed to polymorphism, transmission efficiency, and the historical epidemic development of HIV. The variety of subtypes and sub-subtypes with each having a peculiar three dimensional structure can render a subtype vaccine ineffective for a patient having a different subtype. The high rate of mutation of HIV is certain to complicate selection of the appropriate immunogen.
The preservation of HIV structural components may present performance issues. As with U.S. Pat. No. 5,106,619, both U.S. Pat. Nos. 6,383,806 and 6,503,753 preserve whole particles. The later inventions are directed to inactivating only the RT. The preservation of the antigenic structure is intended to take advantage of a wider range of immunogens. This preservation of the correct antigenic conformation is considered important for access to the cytoplasm via micropinocytosis or mannose-receptor mediated uptake at dendritic cells. U.S. Pat. No. 6,107,543 includes psoralen inactivation within its disclosed method, but conversely required the removal of envelope glycoproteins gp120 and gp160 (but not gp41) because antibodies to those glycoproteins might facilitate virus absorption to cells. In fact, it is known that HIV can bind to and use C3b as ligands to permit infectious immune complexes to bind to dendritic cells and B lymphocytes. Antibodies to gp160 or gp120 sometimes lead to concentrations of virus in the lymph nodes and spleen. The '543 approach, like the others, would preserve transmembrane protein gp41 and some or all of the viral membrane.
At any rate, this preserved viral structure can hold unintended consequences. First, as described above gp160, gp120, and gp41 provide binding sites for complement factor H. (Pinter, Claudia et al, Aids Research and Human Retroviruses, Vol. 11(5), pp. 577-88 (1995) (Pinter, Claudia, et al., Aids Research and Human Retroviruses, Vol. 11(8) (1995))(Stoiber, Heribert, et al., Immunobiology, Vol. 193, pp 98-113 (1995)) Accordingly, the retention of these structures means that factor H will interfere with humoral immune response following vaccination. The removal of gp120 gp160 in U.S. Pat. No. 6,107,543 may mitigate this effect to some degree; nevertheless, the preservation of the gp41 Factor H binding sites would work against the immunogenicity of the composition. Second, both approaches are silent as to the cellular plasma membrane and retain some or all of the viral membrane, including certain bound proteins that interfere with immune response. As an assembling, replicating HIV particle buds through the infected cell plasma membrane, the membrane is enriched by CD55 (the decay accelerating factor) and CD59 (the homologous restriction factor) that regulate complement. These molecules are incorporated into the viral membrane upon budding from infected cells. Preservation of some or all of these features or structures could interfere with complement activation and humoral response. (Saifuddin, 1995) Third, HIV surface components bear sialic acid, which could remain on the preserved structure of inactivated HIV. Sialic acids are typically found on host proteins and cellular structures; high sialic acid content on a virus, even if the virus were inactivated, would limit the host's ability to recognize the virus and respond properly. Importantly, sialic acid residues are also used in the binding of Factor H. (Meri, Seppo, et al., “Discrimination Between Activator and Nonactivators of the Alternative Pathway of complement Regulation: Regulation Via a Sialic acid/Polyanion binding site on Factor H.” Proc. Natl. Acad. Sci., USA, Vol. 87(10), pp. 3982-6 (May 1990)) (Blackmore, T. K., et al., J. of Immunology, Vol. 157(12), pp. 5422-7 (December 1997)) (Kuhn, S., et al., Eur. J. Immunol., Vol. 26(10), pp. 2383-7 (October 1996)) (Pangburn, M. K., et al., J. of Immunology, Vol. 164(9) (May 2000))
The present invention is directed to an immunogenic composition that addresses these issues. It is intended that by creating compounds available to target different subtypes and aspects of HIV, it will advance treatment and research. Ultimately, it is hoped to extend survival and to improve the quality of life for infected individuals.